TABLE 1.
Summary of results for glycine, β-alanine, and taurine-evoked current and fluorescence recordings
Displayed are values for half-maximal activation (EC50), Hill coefficient (nH), maximal current, and fluorescence responses for glycine (DImax and DFmax) and those for β-alanine and taurine, normalized to glycine responses (RI/F = (I/Fmax(b-ala or tau))/(I/Fmax(gly))). †/‡ indicate significant differences to glycine (†) and β-alanine (‡) evoked current EC50 values (p < 0.05, Student's t-test). */# indicate significant differences to maximal glycine (*) and β-alanine (#) evoked current and fluorescence responses (p < 0.05, Student's t-test). n = 3–9 for each experiment. Numbers in superscript indicate the fluorophore used: 1, MTS-TAMRA; 2, MTSR; 3, AF546. All results for fluorescence are shown in bold. Note that all values for glycine-evoked current and fluorescence responses are reproduced from Pless and Lynch (27).
| Construct | Glycine |
β-Alanine |
Taurine |
||||||
|---|---|---|---|---|---|---|---|---|---|
| EC50 | nH | Imax/ΔFmax | EC50 | nH | RI/RF | EC50 | nH | RI/RF | |
| μm | μA/% | μm | μm | ||||||
| WT | |||||||||
| WT ΔI no label | 15.5 ± 0.3 | 2.6 ± 0.1 | 8.3 ± 0.4 | 22.2 ± 0.1† | 2.4 ± 0.1 | 0.97 ± 0.03 | 64.4 ± 1.0†/‡ | 2.2 ± 0.1 | 0.94 ± 0.02 |
| WT ΔI label1 | 15.6 ± 0.4 | 2.7 ± 0.1 | 7.9 ± 0.2 | 20.8 ± 0.2† | 2.5 ± 0.1 | 0.97 ± 0.02 | 60.4 ± 1.5†/‡ | 2.5 ± 0.1 | 0.98 ± 0.02 |
| Loop E | |||||||||
| L127C ΔI1 | 4950 ± 180 | 2.4 ± 0.2 | 7.7 ± 0.8 | 6690 ± 140† | 2.7 ± 0.1 | 1.04 ± 0.10 | 18400 ± 100†/‡ | 2.8 ± 0.1 | 1.00 ± 0.12 |
| L127C ΔF | 6070 ± 240 | 1.8 ± 0.1 | 46.7 ± 7.1 | 47100 ± 3300 | 1.2 ± 0.1 | 2.67 ± 0.35* | 66100 ± 4600 | 1.2 ± 0.1 | 3.19 ± 0.41* |
| Loop D | |||||||||
| Q67C ΔI1 | 63.1 ± 0.6 | 2.9 ± 0.1 | 7.2 ± 0.5 | 240 ± 6† | 2.4 ± 0.1 | 1.11 ± 0.19 | 665 ± 10†/‡ | 2.3 ± 0.1 | 0.92 ± 0.04 |
| Q67C ΔF | 1180 ± 50 | 1.7 ± 0.1 | 18.0 ± 0.6 | 2440 ± 150 | 1.2 ± 0.1 | 7.20 ± 0.78* | 3000 ± 90 | 1.2 ± 0.1 | 11.38 ± 1.50*/# |
| Loop 2 | |||||||||
| A52C ΔI1 | 17.4 ± 0.1 | 3.0 ± 0.1 | 7.7 ± 0.2 | 40.8 ± 0.3† | 2.6 ± 0.1 | 0.85 ± 0.13 | 105 ± 1†/‡ | 2.1 ± 0.1 | 0.99 ± 0.15 |
| A52C ΔF | 201 ± 10 | 1.4 ± 0.1 | −8.6 ± 1.1 | 380 ± 38 | 0.9 ± 0.1 | 0.42 ± 0.02* | 730 ± 76 | 1.1 ± 0.1 | 0.23 ± 0.01*/# |
| Loop F | |||||||||
| V178C ΔI3 | 40.7 ± 0.1 | 2.6 ± 0.1 | 10.3 ± 2.3 | 97.8 ± 9.6† | 1.8 ± 0.3 | 0.85 ± 0.22 | 400 ± 18†/‡ | 1.7 ± 0.1 | 1.11 ± 0.22 |
| V178C ΔF | 313 ± 20 | 1.3 ± 0.1 | 12.6 ± 0.6 | 131 ± 1 | 1.4 ± 0.1 | 0.95 ± 0.15 | 283 ± 19 | 1.3 ± 0.1 | 0.84 ± 0.13 |
| G181C ΔI2 | 39.8 ± 1.3 | 1.9 ± 0.1 | 9.9 ± 2.1 | 121 ± 4† | 2.3 ± 0.2 | 0.95 ± 0.10 | 351 ± 16†/‡ | 1.7 ± 0.1 | 0.92 ± 0.08 |
| G181C ΔF | 503 ± 65 | 1.3 ± 0.2 | 11.6 ± 2.7 | 553 ± 20 | 1.4 ± 0.1 | 0.92 ± 0.24 | 1400 ± 170 | 1.2 ± 0.1 | 0.88 ± 0.19 |
| Loop C | |||||||||
| H201C ΔI1 | 16.3 ± 0.6 | 2.9 ± 0.2 | 9.3 ± 0.8 | 62.3 ± 0.1† | 4.6 ± 0.1 | 1.26 ± 0.07 | 117 ± 3†/‡ | 2.6 ± 0.2 | 0.82 ± 0.07 |
| H201C ΔF | 126 ± 3 | 2.1 ± 0.1 | 10.2 ± 1.0 | 209 ± 39 | 1.3 ± 0.1 | 0.95 ± 0.12 | 365 ± 34 | 1.2 ± 0.1 | 0.78 ± 0.12 |
| N203C ΔI1 | 45.7 ± 1.2 | 3.0 ± 0.2 | 6.9 ± 0.2 | 56.1 ± 1.9† | 3.3 ± 0.2 | 0.93 ± 0.04 | 90.8 ± 4.2†/‡ | 2.6 ± 0.4 | 0.89 ± 0.06 |
| N203C ΔF | 526 ± 25 | 1.2 ± 0.1 | 44.3 ± 5.9 | 228 ± 14 | 0.8 ± 0.1 | 0.92 ± 0.07 | 286 ± 15 | 0.8 ± 0.1 | 0.90 ± 0.14 |
| Pre-M1 | |||||||||
| Q219C ΔI2 | 9.2 ± 0.2 | 2.6 ± 0.2 | 8.4 ± 0.4 | 12.3 ± 1.3 | 2.1 ± 0.4 | 0.86 ± 0.09 | 22.3 ± 0.4†/‡ | 2.6 ± 0.1 | 0.84 ± 0.07 |
| Q219C ΔF | 98.6 ± 9.9 | 1.5 ± 0.2 | −13.3 ± 0.8 | 69.3 ± 5.2 | 1.1 ± 0.1 | 0.90 ± 0.13 | 90.8 ± 5.0 | 1.3 ± 0.1 | 0.81 ± 0.13 |
| M227C ΔI3 | 13.1 ± 0.1 | 2.4 ± 0.1 | 6.5 ± 0.5 | 28.8 ± 0.7† | 2.1 ± 0.1 | 0.79 ± 0.02 | 81.5 ± 1.9†/‡ | 2.0 ± 0.1 | 0.75 ± 0.12 |
| M227C ΔF | 226 ± 24 | 1.0 ± 0.1 | −3.5 ± 0.3 | 98.5 ± 4.6 | 1.1 ± 0.1 | 1.10 ± 0.06 | 336 ± 36 | 1.1 ± 0.1 | 1.13 ± 0.09 |